Isoflavones and compounds derived therefrom (i.e., isoflavonoids) are components unique to leguminous plants and have attracted attention as a health supplement in recent years. In addition, isoflavonoids including isoflavones have been known to play a very important role as antimicrobial agents and symbiotic signals for the plants to adapt to biological environments.
The simplest skeletal structure of isoflavonoid is isoflavone, which is one of a group of isoflavonoids, produced early by flavanoid metabolism (see FIG. 1). Isoflavones and their glycosides are accumulated in organs of leguminous plants. Daidzein (7,4′-dihydroxyisoflavone) and genistein (5,7,4′-trihydroxyisoflavone) contained in the free forms and in the form of glycosides in soybean seeds have been known as phytoesterogen (plant esterogen) for the promotion of the health of humans and for the prevention of diseases.
Isoflavone is an intermediate product in the biosynthesis of isoflavonoids having ecophysiological activity, such as antimicrobial phytoalexins having a pterocarpan or isoflavan skeleton. Approximately 50% of isoflavonoids have functional groups derived from 4′-methoxyl group, and these compounds are mainly derived from 4′-methoxylated isoflavone, formononetin (7-hydroxy-4′-methoxyisoflavone).
The isoflavonoid skeleton is biosynthetically produced from (2S)-flavanone by the action of a cytochrome P450 (P450), i.e., 2-hydroxyisoflavanone synthase (IFS). The IFS catalyzes the hydroxylation of the carbon at position 2 of the flavonoid skeleton with the rearrangement of 1,2-aryl group. The resulting product, 2-hydroxyisoflavanone, is dehydrated to form isoflavone (see FIG. 1).
cDNAs of IFS have been identified in one of the leguminous plants, Glycyrrhiza echinata (hereinafter, referred to as “licorice”) (Non-Patent Document 1 land Patent Document 1) and soybean (Non-Patent Document 2 and Non-Patent Document 3). In the in vitro assay using recombinant IFS which had been overexpressed in yeast microsomes, a large amount of isoflavone was produced by spontanious dehydration of the initial product, 2-hydroxyisoflavanone, in addition to the initial product (Non-Patent Document land Non-Patent Document 3). Furthermore, it was reported that IFS expressed in insect cells produced only isoflavone (Non-Patent Document 2). In this way, an isoflavone can be produced non-enzymatically from the direct product of IFS reaction. In addition, (2S)-flavanone, the substrate of IFS, is a common component present in both leguminous and non-leguminous plants. Thus, it was expected that a non-leguminous plant containing no isoflavonoids could be transformed to the plant having an ability of producing isoflavones by using the IFS cDNA (Non-Patent Document 4, Non-Patent Document 5, and Non-Patent Document 6).
On the basis of these findings, attempts have been made to produce isoflavone in non-leguminous plants (Arabidopsis thaliana and tobacco), which inherently contain no isoflavone, by introduction of a soybean IFS gene. However, the production was as low as around 1/1,000 of that by soybean seeds (Non-Patent Document 3, Non-Patent Document 7, and Non-Patent Document 8). Therefore, it is considered that IFS alone cannot perform the production of isoflavone in an efficient manner.
On the other hand, the enzymatic activity of 2-hydroxyisoflavanone dehydratase, which converts 2,7,4′-trihydroxyisoflavanone into daidzein, was detected in cells of Pueraria lobata (kuzu beans) and the protein thereof was then purified (Non-Patent Document 9 and Non-Patent Document 10). In addition, in experiments conducted by the inventors of the present invention, 2,7-dihydroxy-4′-methoxyisoflavanone was converted into formononetin in licorice cell-free extract but 2,7,4′-trihydroxyisoflavanone was not converted into daidzein (Non-Patent Document 11). These results indicate that the dehydration of 2-hydroxyisoflavanone to isoflavone in plant cells may depend on an enzyme (i.e., 2-hydroxyisoflavaone dehydratase) and the enzyme may have substrate specificity for 2-hydroxyisoflavanones having different substituents.
In this way, the previous studies have revealed that isoflavone cannot be efficiently produced only by IFS and the substrate-specific enzymes play an important role in the dehydration of 2-hydroxyisoflavanones to isoflavones. However, the details of the enzyme (2-hydroxyisoflavanone dehydratase) which contributes to the dehydration of 2-hydroxyisoflavanone, to isoflavones have not been elucidated.
[Patent Document 1]
WO 00/46356 pamphlet
[Non-Patent Document 1]
Akashi, T., Aoki, T. and Ayabe, S. (1999) Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice. Plant Physiol. 121: 821-828.
[Non-Patent Document 2]
Steele, C. L., Gijzen, M., Qutob, D. and Dixon, R. A. (1999) Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch. Biochem. Biophys. 367: 146-150.
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Jung, W., Yu, O., Lau, S. M., O'Keefe, D. P., Odell, J., Fader, G. and McGonigle, B. (2000) Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nature Biotechnol. 18: 208-212.
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Dixon, R. A. and Steele, C. L. (1999) Flavonoids and isoflavonoids—a gold mine for metabolic engineering. Trends Plant Sci. 4: 394-400.
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Humphreys, J. M. and Chapple, C. (2000) Molecular ‘pharming’ with plant P450s. Trends Plant Sci. 5: 271-272.
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Feldmann, K. A. (2001) Cytochrome P450s as genes for crop improvement. Curr. Opin. Plant Biol. 4: 162-167.
[Non-Patent Document 7]
Yu, O., Jung, W., Shi, J., Croes, R. A., Fader, G. M., McGonigle, B. and Odell, J. T. (2000) Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues. Plant Physiol. 124: 781-793.
[Non-Patent Document 8]
Liu, C. J., Blount, J. W., Steele, C. L. and Dixon, R. A. (2002) Bottlenecks formetabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proc. Natl. Acad. Sci. USA 99: 14578-1458 3.
[Non-Patent Document 9]
Sankawa, U. and Hakamatsuka, T. (1997) Biosynthesis of isoflavone and related compounds in tissue cultures of Pueraria lobata. In Dynamic aspects of natural products chemistry. Molecular biological approaches. Edited by Ogura, K. and Sankawa, U. pp. 25-48. Kodansha/Harwood Academic, Tokyo.[Non-Patent Document 10]Hakamatsuka, T., Mori, K., Ishida, S., Ebizuka, Y. and Sankawa, U. (1998) Purification of 2-hydroxyisoflavanone dehydratase from the cell cultures of Pueraria lobata. Phytochemistry 49: 497-505.[Non-Patent Document 11]Akashi, T., Sawada, Y., Aoki, T. and Ayabe, S. (2000) New scheme of the biosynthesis of formononetin involving 2,7,4′-trihydroxyisoflavanone but not daidzein as the methyl acceptor. Biosci. Biotechnol. Biochem. 64:2276-2279.[Non-Patent Document 12]Akashi, T., Sawada, Y., Shimada, N., Sakurai, N., Aoki, T. and Ayabe, S. (2003) cDNA cloning and biochemical characterization of S-adenosyl-L-methionine:2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase, a critical enzyme of the legumes of lavonoid phytoalexin pathway. Plant Cell Physiol. 44:103-112.[Non-Patent Document 13]Ayabe, S., Akashi, T. and Aoki, T. (2002) Cloning of cDNAs encoding P450s in the flavonoid/isoflavonoid pathway from elicited leguminous cell cultures. Methods Enzymol. 357: 360-369.[Non-Patent Document 14]Nakamura, K., Akashi, T., Aoki, T., Kawaguchi, K. and Ayabe, S. (1999). Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract. Biosci. Biotechnol. Biochem. 63: 1618-1620.